Bidirectional electron transfer in photosystem I: electron transfer on the PsaA side is not essential for phototrophic growth in Chlamydomonas

We have used pulsed electron paramagnetic resonance (EPR) measurements of the electron spin polarised (ESP) signals arising from the geminate radical pair P700(z.rad;+)/A(1)(z.rad;-) to detect electron transfer on both the PsaA and PsaB branches of redox cofactors in the photosystem I (PSI) reaction centre of Chlamydomonas reinhardtii. We have also used electron nuclear double resonance (ENDOR) spectroscopy to monitor the electronic structure of the bound phyllosemiquinones on both the PsaA and PsaB polypeptides. Both these spectroscopic assays have been used to analyse the effects of site-directed mutations to the axial ligands of the primary chlorophyll electron acceptor(s) A(0) and the conserved tryptophan in the PsaB phylloquinone (A(1)) binding pocket. Substitution of histidine for the axial ligand methionine on the PsaA branch (PsaA-M684H) blocks electron transfer to the PsaA-branch phylloquinone, and blocks photoaccumulation of the PsaA-branch phyllosemiquinone. However, this does not prevent photoautotrophic growth, indicating that electron transfer via the PsaB branch must take place and is alone sufficient to support growth. The corresponding substitution on the PsaB branch (PsaB-M664H) blocks kinetic electron transfer to the PsaB phylloquinone at 100 K, but does not block the photoaccumulation of the phyllosemiquinone. This transformant is unable to grow photoautotrophically although PsaA-branch electron transfer to and from the phyllosemiquinone is functional, indicating that the B branch of electron transfer may be essential for photoautotrophic growth. Mutation of the conserved tryptophan PsaB-W673 to leucine affects the electronic structure of the PsaB phyllosemiquinone, and also prevents photoautotrophic growth.     

Photoaccumulation of the PsaB phyllosemiquinone in photosystem I of Chlamydomonas reinhardtii

Photoaccumulation of membrane preparations of Chlamydomonas reinhardtii at pH 8 and 220 K reduces the primary and secondary electron acceptors in the Photosystem I (PSI) reaction centre, and produces a maximum of two spins per P700(z.rad;+). Proton electron nuclear double resonance (ENDOR) spectra demonstrate that the phyllosemiquinone produced is that attributed to the PsaA branch of electron transfer. Photoaccumulation at pH 10 and 220 K produces a maximum of four spins per P700(z.rad;+), and proton ENDOR spectra indicate that a second phyllosemiquinone is being photoaccumulated, with markedly different proton hyperfine couplings (hfcs). This phyllosemiquinone is unaffected by mutation of PsaAW693, confirming that it does not arise from the PsaA branch of electron transfer, and we therefore attribute it to the PsaB phyllosemiquinone.     

Photoaccumulation of the PsaB phyllosemiquinone in Photosystem I of Chlamydomonas reinhardtii

Photoaccumulation of membrane preparations of Chlamydomonas reinhardtii at pH 8 and 220 K reduces the primary and secondary electron acceptors in the Photosystem I (PSI) reaction centre, and produces a maximum of two spins per P700⁺. Proton electron nuclear double resonance (ENDOR) spectra demonstrate that the phyllosemiquinone produced is that attributed to the PsaA branch of electron transfer. Photoaccumulation at pH 10 and 220 K produces a maximum of four spins per P700⁺, and proton ENDOR spectra indicate that a second phyllosemiquinone is being photoaccumulated, with markedly different proton hyperfine couplings (hfcs). This phyllosemiquinone is unaffected by mutation of PsaAW693, confirming that it does not arise from the PsaA branch of electron transfer, and we therefore attribute it to the PsaB phyllosemiquinone.     

Oxo-vanadium as a spin probe for the investigation of the metal coordination environment of imidazole glycerol phosphate dehydratase

Imidazole glycerol phosphate dehydratase (IGPD) catalyses the dehydration of imidazole glycerol phosphate to imidazole acetol phosphate, an important late step in the biosynthesis of histidine. IGPD, isolated as a low molecular weight and inactive apo-form, assembles with specific divalent metal cations to form a catalytically active high molecular weight metalloenzyme. Oxo-vanadium ions also assemble the protein into, apparently, the same high molecular weight form but, uniquely, yield a protein without catalytic activity. The VO2+ derivative of IGPD has been investigated by electron paramagnetic resonance (EPR), electron nuclear double resonance (ENDOR) and electron spin echo envelope modulation (ESEEM) spectroscopy. The spin Hamiltonian parameters indicate the presence of multiple 14N nuclei in the inner coordination sphere of VO2+ which is corroborated by ENDOR and ESEEM spectra showing resonances attributable to interactions with 14N nuclei. The isotropic superhyperfine coupling component of about 7 MHz determined by ENDOR is consistent with a nitrogen of coordinated histidine imidazole(s). The ESEEM Fourier-transform spectra further support the notion that the VO2+ substituted enzyme contains inner-sphere nitrogen ligands. The isotropic and anisotropic 14N superhyperfine coupling components are similar to those reported for other equatorially coordinated enzymatic histidine imidazole systems. ESEEM resonances from axial 14N ligands are discussed.     

Bidirectional electron transfer in photosystem I: determination of two distances between P700+ and A1- in spin-correlated radical pairs

The spin-correlated radical pair [P(700)(+)A(1)(-)] gives rise to a characteristic "out-of-phase" electron spin-echo signal. The electron spin-echo envelope modulation (ESEEM) of these signals has been studied in thylakoids prepared from the wild-type strain of Chlamydomonas reinhardtii and in two site-directed mutants, in which the methionine residue which acts as the axial ligand to the chlorin electron acceptor A(0) has been substituted with a histidine either on the PsaA (PsaA-M684H) or the PsaB (PsaB-M664H) reaction center subunits. The analysis of the time domain ESEEM provides information about the spin-spin interaction in the [P(700)(+)A(1)(-)] radical pair, and the values of the dipolar (D) and the exchange (J) interaction can be extracted. From the distance dependence of the dipolar coupling term, the distance between the unpaired electron spin density clouds of the primary donor P(700)(+) and the phyllosemiquinone A(1)(-) can be determined. The [P(700)(+)A(1)(-)] ESEEM spectrum obtained in wild-type thylakoids can be reconstructed using a linear combination of the spectra measured in the PsaA and PsaB A(0) mutants, demonstrating that electron transfer resulting in charge separation is occurring on both the PsaA and PsaB branches. The [P(700)(+)A(1B)(-)] distance in the point dipole approximation in the PsaA-M684H mutant is 24.27 +/- 0.02 A, and the [P(700)(+)A(1A)(-)] distance in the PsaB-M664H mutant is 25.43 +/- 0.01 A. An intermediate value of 25.01 +/- 0.02 A is obtained in the wild-type membranes which exhibit both spin-polarized pairs.     

Evidence for N coordination to Fe in the [2Fe-2S] center in yeast mitochondrial complex III. Comparison with similar findings for analogous bacterial [2Fe-2S] proteins

Yeast mitochondrial complex III contains a subunit with a [2Fe-2S] cluster (the Rieske center) that has unusual physical and chemical properties. For apparently similar centers isolated from bacteria, it has been shown by electron nuclear double resonance (ENDOR) and electron spin echo envelope modulation (ESEEM) measurements that these [2Fe-2S] centers are coordinated by at least one and probably two nitrogen ligands. This work describes similar ENDOR and ESEEM studies on the intact mitochondrial complex. We find that this [2Fe-2S] cluster exhibits ESEEM and ENDOR properties that appear to be indistinguishable from those observed with the isolated bacterial systems. Furthermore, changes in EPR lineshape that occur as complex III is progressively reduced are not accompanied by any changes in the nitrogen coupling parameters. This spectroscopic evidence for nitrogen coordination is supported by published sequence data on four Rieske iron-sulfur subunits. It seems likely that this is a general characteristic of such [2Fe-2S] redox active centers.     

ENDOR and special TRIPLE resonance spectroscopy of photoaccumulated semiquinone electron acceptors in the reaction centers of green sulfur bacteria and heliobacteria.

Photoaccumulation at 205 K in the presence of dithionite produces EPR signals in anaerobically prepared membranes from Chlorobium limicola and Heliobacterium chlorum that resemble the EPR spectrum of phyllosemiquinone (A1*-) photoaccumulated in photosystem I. We have used ENDOR and special TRIPLE resonance spectroscopy to demonstrate conclusively that these signals arise from menasemiquinone electron acceptors reduced by photoaccumulation. Hyperfine couplings to two protons H-bonded to the semiquinone oxygens have been identified by exchange of H. chlorum into D2O, and hyperfine couplings to the methyl group, and the methylene group of the phytyl side chain, of the semiquinone have also been assigned. The electronic structure of these menasemiquinones in these reaction centers is very similar to that of phyllosemiquinone in PSI, and shows a distorted electron spin density distribution relative to that of phyllosemiquinone in vitro. Special TRIPLE resonance spectrometry has been used to investigate the effect of detergents and oxygen on membranes of C. limicola. Triton X-100 and oxygen affect the menaquinone binding site, but n-dodecyl beta-D-maltoside preparations exhibit a relatively unaltered special TRIPLE spectrum for the photoaccumulated menasemiquinone.     

Mutation of the putative hydrogen-bond donor to P700 of photosystem I

The primary electron donor of photosystem I (PS1), called P(700), is a heterodimer of chlorophyll (Chl) a and a'. The crystal structure of photosystem I reveals that the chlorophyll a' (P(A)) could be hydrogen-bonded to the protein via a threonine residue, while the chlorophyll a (P(B)) does not have such a hydrogen bond. To investigate the influence of this hydrogen bond on P(700), PsaA-Thr739 was converted to alanine to remove the H-bond to the 13(1)-keto group of the chlorophyll a' in Chlamydomonas reinhardtii. The PsaA-T739A mutant was capable of assembling active PS1. Furthermore the mutant PS1 contained approximately one chlorophyll a' molecule per reaction center, indicating that P(700) was still a Chl a/a' heterodimer in the mutant. However, the mutation induced several band shifts in the visible P(700)(+) - P(700) absorbance difference spectrum. Redox titration of P(700) revealed a 60 mV decrease in the P(700)/P(700)(+) midpoint potential of the mutant, consistent with loss of a H-bond. Fourier transform infrared (FTIR) spectroscopy indicates that the ground state of P(700) is somewhat modified by mutation of ThrA739 to alanine. Comparison of FTIR difference band shifts upon P(700)(+) formation in WT and mutant PS1 suggests that the mutation modifies the charge distribution over the pigments in the P(700)(+) state, with approximately 14-18% of the positive charge on P(B) in WT being relocated onto P(A) in the mutant. (1)H-electron-nuclear double resonance (ENDOR) analysis of the P(700)(+) cation radical was also consistent with a slight redistribution of spin from the P(B) chlorophyll to P(A), as well as some redistribution of spin within the P(B) chlorophyll. High-field electron paramagnetic resonance (EPR) spectroscopy at 330-GHz was used to resolve the g-tensor of P(700)(+), but no significant differences from wild-type were observed, except for a slight decrease of anisotropy. The mutation did, however, provoke changes in the zero-field splitting parameters of the triplet state of P(700) ((3)P(700)), as determined by EPR. Interestingly, the mutation-induced change in asymmetry of P(700) did not cause an observable change in the directionality of electron transfer within PS1.     

A reaction-induced FT-IR study of cyanobacterial photosystem I.

In oxygenic photosynthesis, photosystem I (PSI) conducts light-driven electron transfer from plastocyanin to ferredoxin. The reactions are initiated when the primary chlorophyll donor, P(700), is photooxidized. P(700) is a chlorophyll dimer ligated by the core subunits psaA and psaB. A difference Fourier transform infrared spectrum, associated with P(700)(+)-minus-P(700), can be acquired using PSI from the cyanobacterium Synechocystis sp. PCC 6803. This spectrum reflects contributions from oxidation-sensitive modes of chlorophyll, as well as from oxidation-induced structural changes in amino acid residues and the peptide backbone. Oxidation-induced structural changes may play a role in the facilitation and control of electron-transfer reactions involving the primary donor. In this paper, we report that photooxidation of P(700) in cyanobacterial PSI perturbs a cysteine residue. At 264 and 80 K, a downshift of a SH stretching vibration from 2560 to 2551 cm(-1) is observed. Such a downshift is consistent with an increase in hydrogen bonding, with a change in C-S-H conformation, or with an electric field effect. Deuterium exchange experiments were also performed. While the perturbed cysteine is in a protein region that is resistant to exchange, other (2)H-sensitive vibrational chl and amino acid bands were observed. From the (2)H exchange experiments, we conclude that photooxidation of P(700) perturbs internal or bound water molecules in PSI and that the P(700)(+)-minus-P(700) spectrum is (2)H exchange-sensitive. The results are consistent with structural complexity in the PSI primary donor, as previously suggested [Kim, S., and Barry, B. A. (2000) J. Am. Chem. Soc. 122, 4980-4981]. Possible explanations, including a partial enolization of P(700)(+), are discussed.     

A new method to determine the structure of the metal environment in metalloproteins: investigation of the prion protein octapeptide repeat Cu2+ complex

Since high-intensity synchrotron radiation is available, extended X-ray absorption fine structure spectroscopy (EXAFS) is used for detailed structural analysis of metal ion environments in proteins. However, the information acquired is often insufficient to obtain an unambiguous picture. ENDOR spectroscopy allows the determination of hydrogen positions around a metal ion. However, again the structural information is limited. In the present study, a method is proposed which combines computations with spectroscopic data from EXAFS, EPR, electron nuclear double resonance (ENDOR) and electron spin echo envelope modulation (ESEEM). From EXAFS a first picture of the nearest coordination shell is derived which has to be compatible with EPR data. Computations are used to select sterically possible structures, from which in turn structures with correct H and N positions are selected by ENDOR and ESEEM measurements. Finally, EXAFS spectra are re-calculated and compared with the experimental data. This procedure was successfully applied for structure determination of the Cu²⁺ complex of the octapeptide repeat of the human prion protein. The structure of this octarepeat complex is rather similar to a pentapeptide complex which was determined by X-ray structure analysis. However, the tryptophan residue has a different orientation: the axial water is on the other side of the Cu.     

Light-induced alteration of low-temperature interprotein electron transfer between photosystem I and flavodoxin

Electron paramagnetic resonance (EPR) was used to study light-induced electron transfer in Photosystem I-flavodoxin complexes. Deuteration of flavodoxin enables the signals of the reduced flavin acceptor and oxidized primary donor, P(700)(+), to be well-resolved at X- and D-band EPR. In dark-adapted samples, photoinitiated interprotein electron transfer does not occur at 5 K. However, for samples prepared in dim light, significant interprotein electron transfer occurs at 5 K and a concomitant loss of the spin-correlated radical pair P(+)A(1A)(-) signal is observed. These results indicate a light-induced reorientation of flavodoxin in the PSI docking site that allows a high quantum yield efficiency for the interprotein electron transfer reaction.     

Contribution of vitamin K1 to the electron spin polarization in spinach photosystem I

The electron spin polarized (ESP) electron paramagnetic resonance (EPR) signal observed in spinach photosystem I (PSI) particles was examined in preparations depleted of vitamin K1 by solvent extraction and following biological reconstitution by the quinone. The ESP EPR signal was not detected in the solvent-extracted PSI sample but was restored upon reconstitution with either protonated or deuterated vitamin K1 under conditions that also restored electron transfer to the terminal PSI acceptors. Reconstitution using deuterated vitamin K1 resulted in a line narrowing of the ESP EPR signal, supporting the conclusion that the ESP EPR signals in the reconstituted samples arise from a radical pair consisting of the oxidized PSI primary donor, P700+, and reduced vitamin K1.     

The function of photosystem I. Quantum chemical insight into the role of tryptophan-quinone interactions

Quantum chemical calculations have provided evidence for the role of tryptophan residues in the electron transfer process of photosystem I (PS-I). The interaction of Trp with quinone acceptors and their radical anions in the A(1) site of PS-I has been modeled by various indole-quinone and indole-semiquinone complexes. MP2 optimizations show that, while neutral quinones and an indole molecule prefer a pi-stacked arrangement, semiquinone radical anions prefer a T-stacked conformation with significant N-H...pi hydrogen bonding interactions. Comparison of density functional calculations of electronic g-tensors with electron paramagnetic resonance data strongly suggests that hydrogen-bonded T-shaped arrangements occur upon reduction of quinone acceptors without an extended side chain (e.g., duroquinone or naphthoquinone), when reconstituted into the phylloquinone-depleted A(1) site of PS-I. In contrast, for the native phylloquinone (vitamin K(1), Q(K)), reorientation of the semiquinone radical anion is prevented by side chain-protein interactions. For a fixed pi-stacked arrangement, the extent of the intermolecular interaction is reduced upon one-electron reduction. This corresponds to a lowering of the redox potential of the P(700)(+)*Q(K)(-)* radical pair, due to interactions of Q(K) with a tryptophan. Together with the comparably weak hydrogen bonding in PS-I, the proposed model explains the very negative redox potential of the A(1) site, needed for forward electron transfer. T-stacking hydrogen bonds to semiquinones may also have to be considered in many other electron transfer processes in living organisms.     

The binding of molybdate to uteroferrin. Hyperfine interactions of the binuclear center with 95Mo, 1H, and 2H

Uteroferrin, an acid phosphatase with a spin-coupled and redox-active binuclear iron center, is paramagnetic in its pink, enzymatically active, mixed-valence (S = 1/2) state. Phosphate, a product and inhibitor of the enzymatic activity of uteroferrin, converts the pink, EPR-active form of the protein to a purple, EPR-silent species. In contrast, molybdate, a tetrahedral oxyanion analog of phosphate, transforms the EPR spectrum of uteroferrin from a rhombic to an axial form. With both electron spin echo envelope modulation (ESEEM) and electron nuclear double resonance (ENDOR) spectroscopies, we observe a hyperfine interaction of [95Mo]molybdate with the S = 1/2, Fe(II)-Fe(III) center of the protein. A pair of 95Mo resonances centered at the 95Mo Larmor frequency at the applied magnetic field and separated by a hyperfine coupling constant of 1.2 MHz is evident. Therefore, a single monomeric species of molybdate is close to, and likely a ligand of, the binuclear cluster. 1H ENDOR studies on uteroferrin reveal at least six sets of lines mirrored about the 1H Larmor frequency. Two pairs of these lines become reduced in intensity when the protein is exchanged against D2O. Moreover, ESEEM and 2H ENDOR spectra display resonances at the 2H Larmor frequency. Therefore, the metal-binding region of the protein is accessible to solvent. Additional deuterium lines observable by ESEEM spectroscopy provide evidence for a population of strongly coupled, readily exchangeable protons associated with the binuclear center. The measured hyperfine coupling constants for these deuterons are orientation-dependent with splittings of nearly 4 MHz at g3 = 1.59 and less than 1 MHz at g1 = 1.94. In the presence of molybdate, ESEEM spectra of D2O-exchanged samples reveal a resonance at the 2H Larmor frequency, with no evidence of spectral components due to strongly coupled deuterons. 1H ENDOR studies of the uteroferrin-molybdate complex show at least seven pairs of lines, mirrored about the 1H Larmor frequency, of which one pair becomes attenuated in amplitude upon deuteration. The active site thus remains accessible to solvent in the presence of molybdate.     

Genetic engineering of thylakoid protein complexes by chloroplast transformation in Chlamydomonas reinhardtii

Chloroplast transformation of Chlamydomonas reinhardtii has developed into a powerful tool for studying the structure, function and assembly of thylakoid protein complexes in a eukaryotic organism. In this article we review the progress that is being made in the development of procedures for efficient chloroplast transformation. This focuses on the development of selectable markers and the use of Chlamydomonas mutants, individually lacking thylakoid protein complexes, as recipients. Chloroplast transformation has now been used to engineer all four major thylakoid protein complexes, photosystem II, photosystem I, cytochrome b ₆/f and ATP synthase. These results are discussed with an emphasis on new insights into assembly and function of these complexes in chloroplasts as compared with their prokaryotic counterparts.Abbreviations ENDOR electron nuclear double resonance - ESEEM electron spin echo envelope modulation - LHC light harvesting complex - PSI Photosystem I - PS II Photosystem II - P₆₈₀ primary electron donor in PS II - P₇₀₀ primary electron donor in PS I     

Radicals associated with the catalytic intermediates of bovine cytochrome c oxidase

Two radicals have been detected previously by electron paramagnetic resonance (EPR) and electron nuclear double resonance (ENDOR) spectroscopies in bovine cytochrome oxidase after reaction with hydrogen peroxide, but no correlation could be made with predicted levels of optically detectable intermediates (P(M), F and F(z.rad;)) that are formed. This work has been extended by optical quantitation of intermediates in the EPR/ENDOR sample tubes, and by comparison with an analysis of intermediates formed by reaction with carbon monoxide in the presence of oxygen. The narrow radical, attributed previously to a porphyrin cation, is detectable at low levels even in untreated oxidase and increases with hydrogen peroxide treatments generally. It is presumed to arise from a side-reaction unrelated to the catalytic intermediates. The broad radical, attributed previously to a tryptophan radical, is observed only in samples with a significant level of F(z.rad;) but when F(z.rad;) is generated with hydrogen peroxide, is always accompanied by the narrow radical. When P(M) is produced at high pH with CO/O(2), no EPR-detectable radicals are formed. Conversion of the CO/O(2)-generated P(M) into F(z.rad;) when pH is lowered is accompanied by the appearance of a broad radical whose ENDOR spectrum corresponds to a tryptophan cation. Quantitation of its EPR intensity indicates that it is around 3% of the level of F(z.rad;) determined optically. It is concluded that low pH causes a change of protonation pattern in P(M) which induces partial electron redistribution and tryptophan cation radical formation in F(z.rad;). These protonation changes may mimic a key step of the proton translocation process.     

Electron spin polarization in photosynthesis and the mechanism of electron transfer in photosystem I. Experimental observations.

Transient electron paramagnetic resonance (EPR) methods are used to examine the spin populations of the light-induced radicals produced in spinach chloroplasts, photosystem I particles, and Chlorella pyrenoidosa. We observe both emission and enhanced absorption within the hyperfine structure of the EPR spectrum of P700+, the photooxidized reaction-center chlorophyll radical (Signal I). By using flow gradients or magnetic fields to orient the chloroplasts in the Zeeman field, we are able to influence both the magnitude and sign of the spin polarization. Identification of the polarized radical and P700+ is consistent with the effects of inhibitors, excitation light intensity and wavelength, redox potential, and fractionation of the membranes. The EPR signal of the polarized P700+ radical displays a 30% narrower line width than P700+ after spin relaxation. This suggests a magnetic interaction between P700+ and its reduced (paramagnetic) acceptor, which leads to a collapse of the P700+ hyperfine structure. Narrowing of the spectrum is evident only in the spectrum of polarized P700+, because prompt electron transfer rapidly separates the radical pair. Evidence of cross-relaxation between the adjacent radicals suggests the existence of an exchange interaction. The results indicate that polarization is produced by a radical pair mechanism between P700+ and the reduced primary acceptor of photosystem I. The orientation dependence of the spin polarization of P700+ is due to the g-tensor anisotropy of the acceptor radical to which it is exchange-coupled. The EPR spectrum of P700+ is virtually isotropic once the adjacent acceptor radical has passed the photoionized electron to a later, more remote acceptor molecule. This interpretation implies that the acceptor radical has g-tensor anisotropy significantly greater than the width of the hyperfine field on P700+ and that the acceptor is oriented with its smallest g-tensor axis along the normal to the thylakoid membranes. Both the ferredoxin-like iron-sulfur centers and the X- species observed directly by EPR at low temperatures have g-tensor anisotropy large enough to produce the observed spin polarization; however, studies on oriented chloroplasts show that the bound ferredoxin centers do not have this orientation of their g tensors. In contrast, X- is aligned with its smallest g-tensor axis predominantly normal to the plane of the thylakoid membranes. This is the same orientation predicted for the acceptor radical based on analysis of the spin polarization of P700+, and indicates that the species responsible for the anisotropy of the polarized P700+ spectrum is probably X-. The dark EPR Signal II is shown to possess anisotropic hyperfine structure (and possibly g-tensor anisotropy), which serves as a good indicator of the extent of membrane alignment.     

Cofactors involved in light-driven charge separation in photosystem I identified by subpicosecond infrared spectroscopy

Photosystem I is one of the key players in the conversion of solar energy into chemical energy. While the chlorophyll dimer P(700) has long been identified as the primary electron donor, the components involved in the primary charge separation process in PSI remain undetermined. Here, we have studied the charge separation dynamics in Photosystem I trimers from Synechococcus elongatus by femtosecond vis-pump/mid-infrared-probe spectroscopy upon excitation at 700, 710, and 715 nm. Because of the high specificity of the infrared region for the redox state and small differences in the molecular structure of pigments, we were able to clearly identify specific marker bands indicating chlorophyll (Chl) oxidation. Magnitudes of chlorophyll cation signals are observed to increase faster than the time resolution of the experiment (~0.2 ps) upon both excitation conditions: 700 nm and selective red excitation. Two models, involving either ultrafast charge separation or charge transfer character of the red pigments in PSI, are discussed to explain this observation. A further increase in the magnitudes of cation signals on a subpicosecond time scale (0.8-1 ps) indicates the formation of the primary radical pair. Evolution in the cation region with time constants of 7 and 40 ps reveals the formation of the secondary radical pair, involving a secondary electron donor. Modeling of the data allows us to extract the spectra of the two radical pairs, which have IR signatures consistent with A+A?- and P???+A?-. We conclude that the cofactor chlorophyll A acts as the primary donor in PSI. The existence of an equilibrium between the two radical pairs we interpret as concerted hole/electron transfer between the pairs of electron donors and acceptors, until after 40 ps, relaxation leads to a full population of the P???+A?. radical pair.     

The contact sites of sickle hemoglobin as seen by spin probe-spin label techniques

The spin-labeled tryptophan (TrpSL) was used as a structural probe of hemoglobin (Hb) contact sites. The electron paramagnetic resonance spectral data indicated that the probe exhibits weak binding to Hb with a dissociation constant of 3.2 x 10(-5) and 4.0 mol bound per Hb tetramer. The spectrum suggested that the bound tryptophan was 'partially immobilized' with a correlation time reflecting the environment of the tryptophan binding site of 8.5 s. The topology of the contact sites was investigated by using a dual spin label methodology in which TrpSL and 2H-15N covalently bound to B 93 cysteine residue were used. The electron spin resonance spectral data suggested that the tryptophan binding sites were located within 8-10 A of the nitroxide free radical of spin-labeled Hb. The environment of the contact sites is discussed.     

Analysis of the spin-polarized electron spin echo of the [P700+ A1-] radical pair of photosystem I indicates that both reaction center subunits are competent in electron transfer in cyanobacteria, green algae, and higher plants

The decay of the light-induced spin-correlated radical pair [P700+ A1-] and the associated electron spin echo envelope modulation (ESEEM) have been studied in either thylakoid membranes, cellular membranes, or purified photosystem I prepared from the wild-type strains of Synechocystis sp. PCC 6803, Chlamydomonas reinhardtii, and Spinaceae oleracea. The decay of the spin-correlated radical pair is described in the wild-type membrane by two exponential components with lifetimes of 2-4 and 16-25 micros. The proportions of the two components can be altered by preillumination of the membranes in the presence of reductant at temperatures lower than 220 K, which leads to the complete reduction of the iron-sulfur electron acceptors F(A), F(B), and F(X) and partial photoaccumulation of the reduced quinone electron acceptor A1A-. The "out-of-phase" (OOP) ESEEM attributed to the [P700+ A1-] radical pair has been investigated in the three species as a function of the preillumination treatment. Values of the dipolar (D) and the exchange (J) interactions were extracted by time-domain fitting of the OOP-ESEEM. The results obtained in the wild-type systems are compared with two site-directed mutants of C. reinhardtii [Santabarbara et al. (2005) Biochemistry 44, 2119-2128], in which the spin-polarized signal on either the PsaA- or PsaB-bound electron transfer pathway is suppressed so that the radical pair formed on each electron transfer branch could be monitored selectively. This comparison indicates that when all of the iron-sulfur centers are oxidized, only the echo modulation associated with the A branch [P700+ A1A-] radical pair is observed. The reduction of the iron-sulfur clusters and the quinone A1 by preillumination treatment induces a shift in the ESEEM frequency. In all of the systems investigated this observation can be interpreted in terms of different proportions of the signal associated with the [P700+ A1A-] and [P700+ A1B-] radical pairs, suggesting that bidirectionality of electron transfer in photosystem I is a common feature of all species rather than being confined to green algae.